The RING ubiquitin E3 ligases are a superfamily of proteins critical to protein homeostasis and signaling in eukaryotes. Dysfunctions in E3 ligases are implicated in innumerable human diseases. This proposal focuses on the regulation of the ubiquitin E3 ligase Parkin. Parkin is central to the controlled destruction of damaged mitochondria by autophagy (mitophagy). Controlled mitophagy is particularly essential to cardiac and neuronal health. Uncontrolled mitophagy due to mutations in Parkin is clearly a driver of early onset Parkinson's disease (eoPD). Parkin is now implicated in a number of other neurological diseases, cardiomyopathy and in various cancers. The central goal here is to create an understanding the physical basis for regulation of Parkin and how clinically observed mutations promote unregulated activity leading to inadequately controlled mitophagy and other biological defects. Though much is known about the biology and structural basis of Parkin function, very little is certain about the physical basis for its regulation. Parkin activity is suppressed by its intra-molecular association with a ubiquitin-like domain and is allosterically activated by the binding of phosphorylated ubiquitin (pUb). Phosphorlyation of the Ubl domain also promotes activation. This complicated intersection of regulatory mechanisms can only be understood by the rigorous dissection of the underlying thermodynamics. Without this knowledge one cannot fully interpret the effects of mutations that lead to disease. We shall take advantage of the broad foundation of knowledge of the biology of Parkin and structural basis of its function to address the poorly understood thermodynamics of allosteric regulation of Parkin. The basis for regulatory control of Parkin will be cast in a modern statistical thermodynamics description of the protein ensemble. The influence of allosteric regulators and post-translational modifications will be examined by comprehensive hydrogen exchange monitored by mass spectrometry and NMR spectroscopy; advanced NMR relaxation techniques; single molecule fluorescence; calorimetry; enzymology; and mutagenesis. A more rigorous and complete understanding of the regulation of Parkin will enable a robust interpretation of pathological mutations. Not all pathological mutations can be simply explained as mutations that disrupt the levels of protein or mutations that directly impact the catalytic site. Examples of common pathological mutations will be examined to reveal the basis for their effects on Parkin's regulatory fidelity, with a longer- range goal of determining how this impact might be mitigated by small molecule intervention.